Abstract

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with generally 4 and 11 N-linked glycans on E1 and E2, respectively. Studies using mutated recombinant HCV envelope glycoproteins incorporated into retroviral pseudoparticles (HCVpp) suggest that some glycans play a role in protein folding, virus entry, and protection against neutralization. The development of a cell culture system producing infectious particles (HCVcc) in hepatoma cells provides an opportunity to characterize the role of these glycans in the context of authentic infectious virions. Here, we used HCVcc in which point mutations were engineered at N-linked glycosylation sites to determine the role of these glycans in the functions of HCV envelope proteins. The mutants were characterized for their effects on virus replication and envelope protein expression as well as on viral particle secretion, infectivity, and sensitivity to neutralizing antibodies. Our results indicate that several glycans play an important role in HCVcc assembly and/or infectivity. Furthermore, our data demonstrate that at least five glycans on E2 (denoted E2N1, E2N2, E2N4, E2N6, and E2N11) strongly reduce the sensitivity of HCVcc to antibody neutralization, with four of them surrounding the CD81 binding site. Altogether, these data indicate that the glycans associated with HCV envelope glycoproteins play roles at different steps of the viral life cycle. They also highlight differences in the effects of glycosylation mutations between the HCVpp and HCVcc systems. Furthermore, these carbohydrates form a "glycan shield" at the surface of the virion, which contributes to the evasion of HCV from the humoral immune response.

Effect of N-glycosylation site mutations on viral-genome replication, E1/E2 expression, and infectious-virion production. (A) Wild-type (WT) and mutated HCV genomes were delivered to CD81-deficient Huh-7w7 cells. Replication was assessed at 72 and 96 h by measuring Renilla luciferase activities in transfected cells. Results are expressed as relative light units (RLU) and are reported as the means ± standard deviations (SDs) of three independent experiments. An assembly-deficient virus (ΔE1E2) and a replication-defective virus (GND) were used as negative controls for assembly and replication, respectively. (B) Forty-eight hours after electroporation of the viral genomes devoid of the luciferase reporter gene, expression of the viral proteins E1, E2, and NS3 was analyzed in cell lysates by Western blotting with specific MAbs (A4 [anti-E1], 3/11 [anti-E2], 486D39 [anti-NS3], and C4 [anti-β-actin]). Mutated E1 and E2 envelope proteins lacking one glycan are indicated as E13g and E210g, respectively. (C) HCVcc produced by Huh-7w7 cells were incubated for 3 h with Huh-7 cells. Luciferase assays were performed on infected cells at 72 h postinfection. Results are expressed as relative light units and are reported as the means ± SDs of three independent experiments. The nonparametric Mann-Whitney test was used to compare the infectivities of the wild-type and mutant HCVcc. Differences were considered statistically significant if P < 0.05 (*).

Effect of N-glycosylation site mutations on HCVcc secretion. (A) Huh-7 cells were transfected, and 72 h posttransfection supernatants were collected. In parallel, virus-producing cells were washed and lysed by repetitive freeze-thaw cycles. Extracellular and intracellular infectivities were determined by measuring Renilla luciferase activities in infected cells. Results are expressed as relative light units and are reported as the means ± SDs of three independent experiments. (B) The results of panel A are expressed as percentages of cell-associated infectivity relative to the total infectivity (intracellular plus extracellular infectivity) and are reported as the means ± SDs of three independent experiments.

Effect of N-glycosylation site mutations on HCVcc assembly. (A and B) Analysis of core release. Mutated HCV genomes were delivered to CD81-deficient Huh-7w7 cells. HCV core release was assessed at 96 h using a fully automated chemiluminescent microparticle immunoassay. Results are expressed as raw data (A) or as percentages of that for the WT (B) and are reported as the means ± SDs of the results of two independent experiments. (C) Infectivity of the glycosylation mutants normalized to the level of core protein secretion. Values obtained for infectious-virion production (Fig. ) have been normalized to those obtained for HCV core release (Fig. ) to compare the specific infectivities of secreted viral particles.

Role of glycans E2N2, E2N4, and E2N7 on genotype 2a HCVpp infectivity. (A) Plasmids encoding the E2N2, E2N4, and E2N7 mutants in the context of a genotype 2a (JFH-1) E1/E2 polyprotein were used to generate HCVpp. Infection assays with the luciferase reporter gene were performed by using target Huh-7 cells. Similar inputs of viral particles were used in each experiment, and this was confirmed by comparing the amounts of capsid protein incorporated into HCVpp (see panel B, anti-CA [CA]). Pseudotyped particles produced in the absence of envelope proteins (ΔE1E2) were used as a control. The results are expressed as percentages of wild-type infectivity (WT) and are reported as means ± SDs of three independent experiments. (B) Incorporation of HCV envelope proteins into HCVpp. Particles were pelleted through 20% sucrose cushions and analyzed by Western blotting. HCV envelope glycoproteins and the capsid protein of murine leukemia virus (MLV) were revealed with the following specific MAbs: anti-E1 (A4), anti-E2 (3/11), and anti-CA (R187). Expression of mutant proteins was verified by direct Western blotting of cell lysates.

Effect of N-glycosylation site mutations on HCVcc sensitivity to neutralization by antibodies purified from HCV-seropositive sera. Neutralization assays were performed by incubating HCVcc glycosylation mutants or WT HCVcc with various concentrations of antibodies (Ab) purified from the sera of patients infected with HCV genotype 1a (A), 1b (B), 2 (C), or 4 (D). After a 2 h-incubation at 37°C, mixtures of HCVcc and antibodies were put into contact with target cells for 3 h. Luciferase assays were performed on infected cells at 48 to 72 h postinfection. Results are expressed as percentages of infectivity relative to infectivity in the absence of antibodies and are reported as the means ± SDs of three independent experiments.

Effect of N-glycosylation site mutations on HCVcc sensitivity to inhibition by MAb 3/11 or cyanovirin-N. Inhibition assays were performed by incubating HCVcc glycosylation mutants or wild-type HCVcc (WT) with various concentrations of MAb 3/11 (A) or cyanovirin-N (CV-N) (B). After a 2 h-incubation at 37°C, mixtures were put into contact with target cells for 3 h. Luciferase assays were performed on infected cells at 48 to 72 h postinfection. Results are expressed as percentages of infectivity relative to infectivity in the absence of inhibitory protein and are reported as the means ± SDs of three independent experiments.

Effect of N-glycosylation site mutations on HCVcc sensitivity to inhibition by CD81-LEL. Inhibition assays were performed by incubating HCVcc glycosylation mutants or wild-type HCVcc (WT) with various concentrations of CD81-LEL. After a 2 h-incubation at 37°C, mixtures were put into contact with target cells for 3 h. Luciferase assays were performed on infected cells at 48 to 72 h postinfection. Results are expressed as percentages of infectivity relative to infectivity in the absence of inhibitory protein and are reported as the means ± SDs of three independent experiments.

Localization of N-linked glycans on the model of HCV glycoprotein E2. The linear sequence of the JFH-1 E2 ectodomain without the stem region is represented as a chain of beads (colored circles) labeled with the corresponding amino acid and threaded onto a class II fold, which is an adapted version of the model recently published by Krey et al. (). The three putative domains are presented in red (DI), yellow (DII), and blue (DIII), and the variable regions (HVR1, HVR2, and IgVR) are indicated in brown. Circles in pale and bright colors represent residues in the background and foreground of the domains, respectively, and are labeled in white and black fonts. Disulfide bonds are indicated by black bars. Glycosylation sites are shown by green circles numbered sequentially. DI domain residues that participate in CD81 binding are outlined in blue. It has been suggested that additional residues in the DIII domain can also affect CD81 binding (). However, mutation of these residues might affect E2 folding, suggesting an indirect role for these residues (). Glycans affecting CD81 binding are highlighted by light green circles.